Various methods have been developed in the prior art to overcome the diffraction limits in microscopy. From WO 2006/127692 or DE 102006021317 A1 a method with the abbreviation PALM (photo activated localization microscopy) is known, which uses a marker substance for displaying a sample, which can be activated e.g., via optic radiation. The marker substance can emit certain fluorescent radiation only in the activated state. Non-activated molecules of the marker substance emit none or at the most no detectable fluorescent radiation, even after exciting radiation impacting them.
Accordingly the activating radiation is generally called the switching signal. In the PALM-method the switching signal is applied such that at least a certain portion of the activated marker molecules are distanced from neighboring activated molecules such that these marker molecules can be separated, measured by the optic resolution of microscopy, or subsequently separated by image processing. Here, it is assumed that a portion of the fluorescence emitters are isolated. After accepting the fluorescent radiation, the center of their distribution of radiation is then determined for these isolated emitters, to a limited extend as caused by the resolution. Based thereupon, by way of calculation the position of the molecules can be determined with higher precision than actually possible based on the optic resolution. This process is called localization. The increased resolution by determining focal points of the distribution of diffraction by way of calculation is also called “super resolution” in the professional English literature. It requires that in the sample at least a portion of the activated marker molecules can be distinguished using optic resolution, thus they are isolated. Then their position can be determined with higher precision; they can be localized.
In order to isolate individual marker molecules the PALM principle uses statistic effects. In a marker molecule, which upon receipt of the switching signal of a predetermined intensity can be excited to emit fluorescent radiation, it can be ensured by adjusting the intensity of the switching signal that the probability to activate marker molecules present in a given area of the sample is so low that there are sufficient sections in which, within the optic resolution, only marker molecules that can be distinguished emit fluorescent radiation.
The PALM principle was further developed with regards to the activation of the molecules to be detected. For example, in molecules showing a long-term non-fluorescent state and a short-term fluorescent state, any separate activation with activating radiation, which can be spectrally distinguished from the exciting radiation, is not required at all. Rather, the sample is first activated with an illuminating radiation of high intensity so that the overwhelming portion of the molecules has been brought into the non-fluorescent long-term state (e.g., a triplet state). The remaining molecules still fluorescing are this way isolated with regards to the optic resolution.
It shall also be mentioned that the PALM-principle has been identified in professional literature with different abbreviations, such as STORM etc. In the present description the abbreviation PALM is used for all microscopic imagery achieving localization exceeding the optic resolution of the device used, by first isolating fluorescent molecules and then localizing them.
The PALM-method is advantageous in that no high localization is required for the illumination. A simple wide-field illumination is possible.
The PALM-principle requires that many individual images are recorded from the sample, each of which respectively including portions of the isolated molecules. In order to display the sample in its entirety the quantity of all individual images must ensure that all molecules have been included in said portions at least once, to the extent possible. Accordingly, the PALM-method regularly requires a plurality of individual images, which leads to a certain duration being necessary for recording an overall image. This leads to considerable computing expenses, because in each individual image a plurality of molecules must be localized by way of calculation. Here, large quantities of data develop.
This precision of localization is achieved only laterally by the localization in the individual images, thus in a single level, which is allocated to the image level of the camera. The methods are therefore restricted in this regard to a two-dimensional sample analysis. Accordingly, the PALM-principle has been combined with TIRF-excitation, which ensures that only fluorophores from a thin layer of the sample are emitting.
One of the important parameters in the PALM-method is the imprecision of localization. It represents a positioning error, by which the respectively detected fluorescence emitter is shown in the final image. This imprecision of localization is a considerable factor, in particular when structures shall be found in this image in a subsequent processing step. The scientific literature has therefore discussed the question from a very early time on, how the precision of localization can be determined. The publication Betzig et al., Science 313, 1642-1645, 2006, allocates a 2D-Gauβ-distribution to each localized fluorescence emitter with regards to the localization, with its standard deviation being equivalent to the determined positioning error. The precision by which a fluorescence emitter can be localized two-dimensionally has been deducted in the publication Thompson et al., Biophysical Journal 82, 2775, 2002, as a function of the pixel size, the photon count, and the intensity of the background radiation (in prior art also called background fluctuation). Further, the imprecision of the localization depends on the final size of the pixel confusion function as well as the pixeling of the sensor. The deduction disclosed in this publication was criticized in prior art as an overly optimistic approximation, i.e. as an approximately predicting localization with insufficient precision. In this regard, reference is made to the publication Williamson et al., Nature Immunology 12, 655, 2011. However it is particularly problematic that the statements made regarding the imprecision of localization can exclusively be used for the two-dimensional localization microscopy.
Accordingly it cannot be used for further developments of localization microscopy, which allows using luminescent marker molecules also in the third spatial direction, representing the depth direction in reference to the display of the sample. For this purpose, approaches are also known from prior art. Here, “depth direction” is understood as the direction longitudinal in reference to the incident light, thus longitudinal in reference to the optic axis.
The publication B. Huang et al., Science 319, page 810, 2008 describes an imagery radiation path for the PALM-principle, in which a weak cylinder lens is located, leading to a targeted astigmatic distortion. In this way the image of the molecule is elliptically distorted on the camera as soon as the molecule is located above or below the focal level, thus the point of symmetry of the pixel confusion function. From the orientation and the extent of distortion the information about the depth location of the luminescent marker molecule can be acquired. A disadvantage of this method lies in the fact that the local environment and the orientation of a molecular dipole may also lead to a distortion of the image of the luminescent marker molecule, not at all connected to the depth location. Such luminescent marker molecules are then allocated to a wrong depth value, depending on their orientation.
The publication Pavani et al., PNAS 106, page 2995, 2009 suggests modifying the pixel confusion function by a spatial phase modulator in the image to form a double helix structure. The pixel images of individual luminescent marker molecules are then coded into double spots, their depth location being in the angular orientation of the joint axis of said double spots.
According to the publication of Shtengel et al., PNAS 106, page 3125, 2009 photons emitted by the luminescent marker molecules interfere with themselves. For this purpose two lenses assembled in a 4π-configuration are used, which simultaneously observe the luminescent marker molecules. The partial radiation paths obtained in this way are made to interfere via a particular three-path beam splitter. Each of the images obtained here is detected by a camera. The intensity ratios of the images allow conclusions about the depth location.
The publications Toprak et al., Nanolet. 7, pages 3285-3290, 2007 as well as Juette et al., Nature Methods 5, page 527, 2008, describe an approach in which a 50/50 beam splitter is installed in the imaging radiation path, which splits the image of the sample into two partial images. These two images are detected independently.
Additionally, in one of the partial radiation paths obtained here, an optic path length difference is introduced such that two object levels result from the two partial radiation paths, which are separated from each other, for example, by a half or an entire optic minimum resolution (for example 700 nm) in the z-direction, thus the depth direction. The depth position of marker molecules located between these two levels can now be determined by analyzing the two partial images of the same marker molecule (e.g., with regards to the width of the pixel confusion image) or by appropriate fitting of a three-dimensional pixel confusion function. The method requires two partial high-resolution images and a precise adjusting of the radiation paths and calibration measurements in order to achieve a sub-pixel precise interference of the two partial images. Further, the two partial images of a marker molecule generally show a different form because the lateral extension of the pixel confusion function of a displaying system changes depending on the location of the observed object level.
DE 102009060490 A1, which for the rest also lists further literature references regarding 3D-high resolution, also follows the generic approach according to Toprak et al., i.e. to split the image of the sample into two partial images.
Additional literature for high-resolution localization microscopy and particularly also 3D-localization is found in: Baddeley et al., Microscopy & Microanalysis 16, 64, 2010; Baddeley et al., PlosOne 6, e20645, 2011; Juette et al., Nature Methods 5, 527, 2008, Mlodzianoski et al., Optics Express 19, 15009, 2011; Mortensen et al., Nature Methods 7, 377, 2010; Owen et al., Journal of BioPhotonics 3, 446, 2010.